The present invention is generally in the area of gene therapy, and especially prolonged delivery to and expression of enzymes in the brain.
Safe and effective methods for gene delivery into CNS neurons is necessary for successful genetic therapy of neurogenetic disorders. A large number of both viral and non-viral systems have been used for gene transfer into neurons both in vitro and in vivo. The most efficient systems for gene transfer are vectors based on viruses, most notably Herpes Simplex Virus (HSV) (Geller et al. 1995, During et al. 1994), Adenovirus (Davidson et al. 1993; La Gal La Salle 1993), Adeno-associated Virus (AAV) (Kaplitt and During, 1995; During and Leone, 1996) and Lentiviruses (Naldini et al. 1996).
Each of these systems have specific advantages based on efficiency of transduction, packaging capability, neurotropism and stability of expression but all share problems with varying degrees of inflammatory responses, potential for recombination and/or helper virus contamination and replication competency. These issues of safety have precluded the use of any these systems for neuronal gene transfer in humans. Alternative, potentially safer approaches for brain gene delivery include the use of naked, plasmid DNA as well as liposome-DNA complexes (Ulrich et al., 1996; Gao and Huang, 1995). Plasmid DNA appears efficient for transducing muscle (Davis et al., 1993) and liver (following intraportal administration, Wolf, personal communication). However, direct injection of plasmid DNA into the brain is very inefficient. Liposome-DNA complexes are perhaps a little more efficient than naked DNA but compared to viral vectors remain inefficient. Moreover gene transfer is generally transient (Roessler and Davidson, 1994; Kozarsdy and Wilson, 1993).
The major limitations facing current gene therapy is the lack of transduction efficiency and the inherent toxicity and immunogenicity of the most commonly-used vectors. Synthetic supramolecular complexes may provide for safe and effective gene delivery vehicles which could be used in the clinic. The specific problems that gene transfer in the brain present will require both improvements in the. vector system as well as delivery methods for global gene transfer.
A major limitation with brain gene transfer relates not only to the inefficiency of transduction of post-mitotic neurons and glia but also the inaccessibility of the brain. A problem facing neurological gene therapy is the need for global delivery and efficient transduction of cells throughout the brain for most neurogenetic disorders. Viral vectors do not diffuse well through brain parenchyma (Doran et al., 1995), perhaps reflecting size and affinity for cell membrane proteins. A variety of approaches have been used for global gene delivery. These include multiple stereotactic injections (Boviatsis et al., 1994); convection approaches (Oldfield et al., 1994) and hyperosmotic permeation of the blood-brain-barrier (bbb) (Neuwelt et al., 1994). Each of these methods is associated with significant morbidity, moreover the efficacy of each method is limited with bbb permeation effective for drug and gene deliver to tumors, which already have an impaired blood brain barrier, but very poor penetration into normal brain parenchyma.
It is therefore an object of the present invention to provide a means for improved delivery and expression of gene therapy, especially in the brain.
A gene delivery system which is both safe and results in long-term expression has been developed. The method yields widespread gene delivery throughout the brain. A lipid-entrapped, polycation-condensed DNA (LPD) system has been developed for brain gene delivery, using an adeno-associated viral (xe2x80x9cAAVxe2x80x9d) vector in which the transcription unit is flanked by the 145 bp inverted terminal repeats (ITR) of the adeno-associated virus. This AAV plasmid is more effective than a non-ITR containing plasmid in vivo. In addition, an intraventricular delivery system has been developed to obtain widespread, global delivery.
The results show that the LPD-AAV plasmid complexes efficiently transduce neurons and that gene expression can persist for over 10 months in the rat brain. Furthermore, the intraventricular delivery method with systemic hyperosmolality results in global gene delivery. The examples show that expression of the human aspartoacyclase (xe2x80x9cASPAxe2x80x9d) gene can be obtained over a prolonged period of time. Trials in children with this metabolic disorder show that gene expression can be obtained over a period of many months to a year, with functional activity.
A gene delivery system which is both safe and results in long-term expression has been developed. Furthermore, a method to obtain widespread gene delivery throughout the brain has been developed.
Lipid Carriers
A lipid-entrapped, polycation-condensed DNA (LPD) system for brain gene delivery has been developed. The LPD particles are small, about 60-80 nm, monodispersed and colloidally stable (Gao and Huang, 1996). These particles have been shown in vitro to be highly effective in transduction efficiency (approximating that of adenoviral vectors) and safe (Huang, 1997).
The LPD complexes represent a significant step forward in non-viral, synthetic delivery systems:
1) The CD-Chol/DOPE liposome backbone is safe and has been used in 2 published clinical trials (Kaplen et al., 1995; Nabel et al., 1993) and 3 other ongoing protocols.
2) Both poly-L-lysine and protamine have been used clinically and appear safe with protamine being widely used as a heparin antagonist clinically.
3) AAV plasmids have previously been shown to enhance transduction efficiency and stability (Vieweg et al., 1995; Philip et al., 1994).
4) Both prolamine and poly-l-lysine have nuclear localization signals which may also improve transduction efficiencies.
5) Both protamine and poly-l-lysine efficiently complex plasmid DNA and result in condensation of the DNA. This condensed DNA changes from an external diameter of approximately 200nm to a 50-100 nm size when further complexed with the cationic liposome. These nano-LPD particles efficiently transduce post-mitotic cells including neurons and glia.
Other carriers such as liposomes, polycationic complexes and polymeric carriers can also be used, but are not preferred.
AAV Vectors
The gene to be delivered is placed into an adeno-associated viral vector. In the preferred embodiment, the transcription unit is flanked by the 145 bp inverted terminal repeats (ITR) of the adeno-associated virus. These ITRs have been shown to both enhance and prolong gene expression in both prostatic and lympocytic cell lines in vitro (Vieweg J, et al., 1995; Philip R, et al, 1994). The vector also includes a CMV promoter. This AAV plasmid is more effective than a non-ITR containing plasmid in vivo.
Other viral vectors can also be used, including retroviral vectors and adenoviral vectors, although these are not preferred for regulatory issues.
Means for Administration
In the preferred application for this technology, the viral vector is administered to the brain using a syringe or catheter. Intraventricular delivery is used to obtain widespread, global delivery.
Uptake is enhanced using agents such as mannitol, which cause a systemic hyperosmolality. For example, intraparenchymal penetration is enhanced by lowering brain interstitial pressure by the use of systemic mannitol (Rosenberg G A, et al., 1980).